“DNA sequencing” generally refers to methodologies aiming to determine the primary sequence information in a given nucleic acid molecule. Traditionally, Maxam-Gilbert and Sanger sequencing methodologies have been applied successfully for several decades, as well as a pyrosequencing method. However, these methodologies have been difficult to multiplex, as they require a wealth of labor and equipment time, and the cost of sequencing is excessive for entire genomes. These methodologies required each nucleic acid target molecule to be individually processed, the steps including, e.g., subcloning and transformation into E. coli bacteria, extraction, purification, amplification, and sequencing reaction preparation and analysis.
So called “next-generation” technologies or “massive parallel sequencing” platforms allow millions of nucleic acid molecules to be sequenced simultaneously. The methods rely on sequencing-by-synthesis approach, while certain other platforms are based on sequencing-by-ligation technology. Although very efficient, all of these new technologies rely on multiplication of the sequencing templates. Thus, for each application, a pool of sequencing templates needs to be produced. A major advancement for template generation was the use of in vitro transposition technology. The earliest in vitro transposition-assisted sequencing template generation methodology (Tenkanen U.S. Pat. No. 6,593,113) discloses a method in which the transposition reaction results in fragmentation of the target DNA, and the subsequent amplification reaction is carried out in the presence of a fixed primer complementary to the known sequence of the target DNA and a selective primer having a complementary sequence to the end of a transposon DNA.
In vitro transposition methodology has also been applied to “next generation” sequencing platforms. Grunenwald (U.S. Patent Application 20100120098) disclose methods using a transposase and a transposon end for generating extensive fragmentation and 5′-tagging of double-stranded target DNA in vitro. The method is based on the use of a DNA polymerase for generating 5′- and 3′-tagged single-stranded DNA fragments after fragmentation without performing a PCR amplification reaction.
Many “next-generation” sequencing instruments require a specific calibration sequence to be read first as a part of the sequence to be analyzed (e.g. ion torrent PGM and Roche 454 Genome Sequencer FLX System). This calibration sequence has known bases in particular order and it calibrates the instrument so that it is capable of differentiating the signal generated from different bases during the DNA sequencing reaction. It is necessary that each of the sequencing templates comprises this calibration sequence.
Methods that facilitate the downstream handling of the fragmented DNA obtained from the transposition step are needed.